Benefit of glucosyl Hesperidin in patients with primary biliary cholangitis: A multicenter, open-label, randomized control study

Introduction: Globally, the number of patients with primary biliary cholangitis (PBC) is increasing. Growing evidence suggests that oxidative stress plays a significant role in the pathogenesis of chronic liver disease regardless of its etiology. Hesperidin, a natural antioxidative substance derived from citrus peel, has been shown to have an anti-inflammatory effect in a rat arthritis model and may be a potential substance to attenuate intrahepatic inflammation in patients with PBC. In this study, the potential of glucosyl hesperidin as a therapeutic agent for PBC will be investigated through antioxidative stress mechanisms. Methods: Patients with PBC who are 20 years or older will be eligible to participate. Patients will be assigned to 1 of 2 groups and given either 500 or 1000 mg of glucosyl hesperidin per day. The primary endpoint is the ratio of changes in serum gamma-glutamyl transferase levels before and after 24 weeks of glucosyl hesperidin administration. The secondary endpoints are serum hepatobiliary enzyme levels (alkaline phosphatase, transaminase, and total bilirubin levels) and the protein expression levels of nuclear factor erythroid 2-related factor 2 and its target molecule 8, 16, and 24 weeks after administration compared to before administration. Discussion: The prospective clinical interventional study was designed to assess the supportive effect of glucosyl hesperidin on hepatic function in patients with PBC receiving basic ursodeoxycholic acid treatment.


Introduction
In Japan, the number of patients with chronic liver disease has risen to 9 million for chronic hepatitis and 0.4 to 0.5 million for liver cirrhosis. [1] The etiology of liver cirrhosis is changing, owing primarily to the establishment of chronic hepatitis C treatment, and the proportion of non-viral liver diseases is actually increasing. In fact, the number of patients with autoimmune hepatitis and/or primary biliary cholangitis (PBC) has more than tripled in the last decade. [2] Even though treatments for viral hepatitis have made significant progress, the pathophysiology of autoimmune hepatitis and PBC remains unknown, and no curative treatment has been established.
Growing evidence suggests that oxidative stress plays a significant role in the pathogenesis of chronic liver disease regardless of the cause of liver damage. [3][4][5][6][7] Furthermore, in patients with PBC, a mechanism that impairs antioxidant activity via the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 pathway was reported. [8] However, it is unknown whether activating the Nrf2/Keap1 pathway improves PBC pathology.
Ursodeoxycholic acid (UDCA) is widely used as a basic therapeutic agent for PBC and has been reported to promote hepatic Nrf2 activation and reduce oxidative stress. [9] However, because intact enterohepatic circulation is required for its medicinal effect, its effect is significantly attenuated in the pathological condition in which cholestasis has progressed when compared to the early stage of the disease. [10] Previous research has shown that oxidative stress is an independent predictor of stage progression in patients with PBC [11] and that antioxidant levels of vitamins A, C, and E are significantly lower. [12] Taking these facts into account, Nrf2 activation was linked to an improvement in the pathological condition of PBC.
Glucosyl hesperidin is a type of water-soluble polyphenol with high component stability among antioxidants, and its effect is expected to be sufficient. It has been certified by Japanese Agricultural Standards as a safe anti-whitening agent for canned oranges or orange juice. Glucosyl hesperidin is a water-soluble derivative of hesperidin, and hesperidin was found in sera hydrolyzed with beta-glucuronidase after the oral administration of glucosyl hesperidin. [13] Serum hesperidin levels increase in a dose-dependent manner without causing any adverse events (AEs), and human blood levels have been reported to be approximately 12 µM in the case of a 3000 mg oral intake. [14] In this study, hesperidin was effective when administered with standard anti-rheumatoid therapy in ameliorating rheumatoid arthritis in mice and humans without any adverse effects. In a rat study, hesperidin oral administration resulted in concentration-dependent activation of Nrf2 in the liver and improvement of the inflammatory index. [15] Additionally, organ protective effects of hesperidin on liver and spleen were also rereported. [16][17][18] By the way, there have been many reports which show that improvement over time in biochemical test values such as serum gamma-glutamyl transferase (GGT) and alkaline phosphatase after medical therapy intervention is very useful as a better indicator of long-term life prognosis in patients with PBC. [19][20][21][22] Based on these evidences, the potential of glucosyl hesperidin as a therapeutic agent for PBC will be investigated in this study through antioxidative stress mechanisms by evaluating the improvements of hepatic biochemical indicis including GGT and alkaline phosphatase.

Design
This is a multicenter, open-label, randomized, parallel-group study comparing the influence of glucosyl hesperidin on liver function with UDCA treatment in patients with PBC ( Fig. 1). Patients aged 20 years or older are eligible to participate. The inclusion and exclusion criteria are listed in Table 1. Patients will be randomly assigned to either the 500 mg/day or 1000 mg/day treatment groups. In these treatment groups, either 500 mg or 1000 mg of glucosyl hesperidin will be given orally once per day for 24 weeks in addition to ongoing UDCA treatment.
This study will be carried out in accordance with the Helsinki declaration (revised in October 2013) and the Clinical Trials Act of the Japanese Ministry of Health, Labour, and Welfare. The investigator will provide each patient with a thorough explanation of the study and will obtain written informed consent. The study protocol was approved by a certified ethics committee. The research will last from March 30, 2022, to March 31, 2024. The study's organization is shown in jRCTs051210210.

Randomization
The study participants will be registered and allocated using stratified permuted block randomization. Participants will be randomly assigned to either the 500 mg/day or 1000 mg/day groups based on the following factors: registered facility and patient sex. Table 2 provides an overview of all visit and test schedules.

Endpoints
The primary endpoint is the rate of change in serum GGT levels from 0 to 24 weeks after glucosyl hesperidin administration. AEs will be collected for safety analysis. Any unfavorable or unexpected sign, symptom, or disease, including abnormal laboratory results, is considered an AE. Patients who have had their safety evaluation data collected after the start of study treatment will be included in the safety analysis set. The full analysis set and per-protocol set will be used to assess efficacy. The full analysis set is defined as the patient population that was excluded from the safety analysis set for any of the following reasons: ineligible cases after enrollment; and cases with no efficacy data at any time after the start of study treatment. Figure 1. Study design. A total of 110 patients with PBC (55 in each group) will participate in this study. Following eligibility confirmation, patients will be randomly assigned to either the hesperidin 500 mg or 1000 mg treatment groups by an envelope method.

Sample size
Since PBC, the study's target disease, is a rare disease, the total number of participants in the study's participating facility is expected to be around 110, and it is estimated to be 100 after the dropout. The difference between the treatment groups (standard and high dose administration groups) from the start (0 weeks) to the end of administration (24 weeks) is 5%, and the standard deviation is 5%. With a 7.5% probability, a power of 90% can be obtained at a significance level (both sides) of 5%.

Statistical analysis
2.5.1. Collection of background (baseline) data. The background (baseline) data of the research subjects will be tabulated. For nominal and ordinal variables, the frequency and proportion of categories will be calculated. For continuous variables, summary statistics such as mean, standard deviation, minimum, median, and maximum will be calculated. Values of P < .05 will be considered as statistical significance. JMP version 14.3 (SAS Institute Inc, Cary, NC) software and EZR software, which is a GUI for the R statistical computing and graphics environment, [23] will be used for statistical analyses.

Primary endpoint.
The average "rate of change of biliary enzyme levels (serum GGT levels) at 24 weeks after glucosyl hesperidin administration from before administration (0 weeks)" will be compared between groups using an unpaired Student's t test. In addition, the point estimate of the difference between the groups and the 95% confidence interval will be calculated.
The rate of change will be calculated as follows: serum GGT level (24 weeks)-serum GGT level (0 weeks)/serum GGT level (0 weeks).

Secondary endpoint.
For each item, the mean values for each group will be compared between groups using an unpaired Student's t test. In addition, the point estimate of the difference between the groups and the 95% confidence interval will be calculated. The rate of change will be calculated as follows: each evaluation item (at each evaluation point other than 0 weeks)each evaluation item (0 weeks)/each evaluation item (0 weeks). The following items will be included: Rate of change in biliary enzyme levels (serum GGT level) after glucosyl hesperidin administration (8 and 16 weeks) from before administration (0 weeks); rate of change in hepatobiliary enzyme levels (serum alkaline phosphatase, transaminase, and total bilirubin levels) after glucosyl hesperidin administration (8,16, and 24 weeks) from before administration (0 weeks); and rate of change in Nrf2 protein expression levels and its target gene (HMOX-1) after glucosyl hesperidin administration (8,16, and 24 weeks) from before administration (0 weeks).

Exploratory endpoint.
For each item, the mean values for each group will be compared between groups using an unpaired Student's t test. In addition, the point estimate of the difference between the groups and the 95% confidence interval will be calculated. The rate of change will be calculated as follows: each evaluation item (at each evaluation point other than 0 weeks)-each evaluation item (0 weeks)/each evaluation item (0 weeks).
The following items will be included: The rate of change in the protein expression level of inflammatory cytokines (tumor necrosis factor-alpha, interleukin 6, and IL-1β), as well as peroxisome proliferator-activated receptor-gamma (peroxisome proliferator-activated receptor-γ), superoxide dismutase, and glutathione peroxidase, which have an inhibitory effect on the activation of the inflammation/immune-related transcription factor nuclear factor-kappa B before and after glucosyl hesperidin administration (0-8, 0-16, and 0-24 weeks).  2.5.5. Other analysis items. The ratio for each research subject and the average will be calculated to determine adherence. In particular, the previous dose intake rate corresponds to: 70% or more, 30% or more and less than 70%, or less than 30% for each visit during the period of glucosyl hesperidin administration.
2.5.6. Safety analysis. All AEs will be evaluated according to the Common Terminology Criteria for Adverse Events version 5.0 proposed by National Cancer Institute. The incidence of each AE will be calculated in the glucosyl hesperidin-administered subjects during the study's observation period. The period for collecting data related to all evaluation items will be completed for all analyses, and the analysis will be performed once the data is fixed.
2.5.7. Data monitoring. Serum levels of GGT, alkaline phosphatase, transaminase, and total bilirubin will be measured in the clinical laboratories in each facility. Those of tumor necrosis factor-alpha, interleukin 6, IL-1β, peroxisome proliferatoractivated receptor-γ, superoxide dismutase, glutathione peroxidase, Nrf2, and HMOX-1 will be measured by the authors using the commercially available enzyme-linked immunosorbent assay kit. All data will be stored in a database in a locked cabinet at the laboratory of the Department of Gastroenterology and Hepatology at Nara Medical University. The assessors will enter the data into the database system. The trial enrollment and duration will be limited to ensure data robustness.
2.5.8. The quality and safety monitoring. The trial's safety will be monitored once a month by an independent physician with relevant expertise. All participants and visits will be monitored.
On the other hand, the auditors will inspect the process several times throughout the period.

Confidential.
All personal information about potential and enrolled participants, as well as proxies, will be kept in a secure location to which no third party will have access.

Discussion
This study was designed to assess whether glucosyl hesperidin has a positive effect on hepatic function in patients with PBC. Patients with PBC, who are mostly middle-aged and older women, have a relatively high prevalence of comorbidities and are generally prone to high oxidative stress conditions. In addition, previous studies have shown that antioxidant activity via the Nrf2/Keap1 pathway is more severely impaired with the exacerbation of liver pathology in patients with PBC. [8] Furthermore, it has recently been reported that S-adenosyl-Lmethionine, which is associated with redox regulation, protects against mitochondria-derived oxidative stress by upregulating the expression of Nrf2 and its target gene, HMOX-1, in human cholangiocytes. [24] Thus, the Nrf2/Keap1 pathway is generally impaired in chronic liver diseases, including PBC, and attempts to inhibit the pathological progression of chronic liver diseases by activating this pathway are easily understood. UDCA, the first-line drug for PBC, has the ability to activate the Nrf2/ Keap1 pathway. However, because enterohepatic circulation is required for its efficacy, UDCA's pharmacological effect naturally diminishes as intrahepatic biliary stasis progresses. Therefore, new drug candidates for the treatment of PBC must include ingredients that are safe to use in combination with UDCA in terms of duration and dosage. In this regard, glucosyl hesperidin has been chosen as the target component of this medical intervention study, and a new treatment regimen combining UDCA, the standard treatment for PBC, with glucosyl hesperidin has been proposed, with a randomized clinical trial to confirm its efficacy. Glucosyl hesperidin is a common natural component derived from citrus plants that acts as a safe and effective Nrf2/Keap1 pathway activator. [25] The concept of using an existing chemical substance with established safety for oral intake to improve the condition of a designated intractable disease whose pathology is not fully understood is novel and will contribute to the development of a new treatment option. If this study shows that glucosyl hesperidin has a dose-dependent effect on lowering serum biliary enzyme levels, it would be very significant because it would mean that intrahepatic microcholangitis in patients with PBC is being treated, and thus may contribute to the suppression of liver fibrosis progression in a physically safe manner. Furthermore, if the medical efficacy of glucosyl hesperidin in patients with PBC is confirmed, the impact of these study's findings will be enormous, as it may lead to improvements in the pathogenesis of numerous other inflammatory diseases in which disruption of the antioxidant stress mechanism is a major etiological factor. In this study, case enrollment began in the summer of 2022, with results expected in 2024.